Polyethylene glycols (hereinafter also referred to as “PEG”) are compounds that are inactive in the living body. It has been known that the stability and pharmacokinetic properties of proteins and liposomes in the living body can be improved by modifying them with PEG. Proteins modified with PEG and liposomes whose retentivity blood is enhanced by coating them with PEG have already entered the phase of commercialization pharmaceutical products. However, it has been reported that after long-term bolus injection of PEGylated proteins in animal trials, PEG accumulation in the kidney and kidney cavitation occurred. Particularly speaking, PEG is not a biodegradable polymer and has various problems yet to be clarified, including its accumulation and safety in the body after long-term injection to humans. Further, in recent years, some reports have appeared about the phenomenon causing PEG conjugates and PEGylated liposomes to be cleared unusually rapidly after their second dose (accerelated blood clearance phenomenon; hereinafter also referred to as “ABC phenomenon”) (Non-Patent Documents 1 and 2); thus, it cannot be said that the safety and efficacy of PEGylated pharmaceutical products has been fully established.
In the meantime, there is a polymer that has already been used in clinical settings—hyaluronic acid. Hyaluronic acid (hereinafter also referred to as “HA”) is a polysaccharide that was first isolated from the vitreous humour of the bovine eye by K. Meyer in 1934, and has long been known as a major component of the extracellular matrix. HA is a type of glycosaminoglycans composed of disaccharide repeating units of D-glucuronic acid and N-acetylglucosamine linked by β-(1→3) glycosidic bonds. HA has no species difference in its chemical or physical structure, and the HA metabolic system exists even in humans. Hyaluronic acid is also known as a very safe biomaterial from the viewpoint of immunity or toxicity.
In recent years, hyaluronic acid has attracted attention from the viewpoint of not only its safety, as mentioned above, but also the roles that it plays as a physiologically active substance in cell adhesion, cell growth, and induction of cell migration. As regards its production, the mass production of high-molecular-weight hyaluronic acid using microorganisms has been succeeded, and intensive research has been made on drug delivery systems (hereinafter also referred to as “DDS”) using hyaluronic acid. There were other reports stating that conjugation of hyaluronic acid to a drug makes it possible to achieve targeting of the drug to cancer tissues (Patent Document 1), targeting of the drug to the liver (Patent Document 2), and reduction in antigenicity (Patent Document 3).
The drawback of hyaluronic acid when being used as a DDS substrate for targeting or extention in retension time is its poor retentivity in blood. Six consecutive saccharides are believed to be the site of hyaluronic acid that is recognized by the receptor, and various attempts have been made to elongate the retention time of HA in blood by modifying its carboxy groups (Patent Document 4, 5 and 6).
There was developed a hyaluronic acid derivative whose retentivity in blood was increased by highly modifying carboxy in the glucuronic acid moiety of hyaluronic acid, with its usefulness being demonstrated (Patent Document 7). In general, the retentivity of a hyaluronic acid derivative in blood is elongated by increasing the modification degree of carboxy in its glucuronic acid moiety. However, it has been found that both factors do not linearly correlate with each other but the correlation dramatically changes once the particular threshold is exceeded.
Oligonucleotides such as antisense DNA/RNA and siRNA, which have been being developed as nucleic acid pharmaceuticals in recent years, are subject to degradation by nucleases in and outside the living body, are rapidly degraded when they are intravenously (hereinafter also referred to as “iv”) injected alone. In order for nucleic acid pharmaceuticals to display their efficacy, it is essential to deliver the nucleic acids into the cytoplasm or nucleus. A majority of conventional pharmaceutical products comprising proteins or peptides as an active ingredient act on extracellular targets. In order to develop more innovative pharmaceutical products, a means to allow proteins or peptides to act on intracellular targets is needed. For this purpose, there is a need to develop a process for delivering proteins or peptides into the cytoplasm, more specifically a process by which after a medicament is taken up by the cell via endocytosis, the active ingredient of the medicament is effectively released from the endosome into the cytoplasm.
Synthetic canonic polymers make it possible to electrostatically condense the negatively charged gene and deliver the gene into the cytoplasm and, thus, have been considered as effective as a gene carrier. The synthetic cationic polymers that have been reported include poly-L-lysine (Patent Document 8, Non-Patent Document 3), polyethyleneimine (Non-Patent Document 4), a synthetic polymer having an imidazolyl group (Non-Patent Document 5), and a polyamidoamine dendrimer (Non-Patent Document 6). In particular, it has been shown that the polyethyleneimine having secondary amine and the synthetic polymer having an imidazolyl group are taken up into the cytoplasm with high transfer efficiency due to their proton sponge effect. However, these synthetic polymers are polyamines that are generally believed to have high cytotoxicity, and are not completely ensured to be safe. There was another example of using chitosan, a cationic polysaccharide polymer (Non-Patent Document 7), but this technique has not yet been put to practical use because of its low gene transfer efficiency. Still another report was made on an attempt for intracytoplasmic transfer of siRNA using a nanogel that is obtained by modifying carboxy in hyaluronic acid with a compound having a mercapto group (thiol) and ultrasonically crosslinking the modified HA (Non-Patent Document 17).
There were also various reports about intracytoplasmic transfer of proteins/peptides. Examples include the reports on attempts for intracytoplasmic transfer through the modification of a peptide with a cell-penetrating peptide (cpp) (Non-Patent Document 8) or through the formation of a protein/peptide complex comprising a cationic libosome as a carrier (Non-Patent Document 9), but the transfer efficiencies of these techniques were not necessarily high.
Further, in order to reduce cytotoxicity, various approaches were attempted, including PEGylation of a polyamine or modification of HA with a polyamine (Non-Patent Document 10), and modification of a polyamine with a functional group that detaches in a pH-responsive fashion (e.g., at low pH). However, polyamines are inherently used even in these approaches, so further work and studies are needed on the toxicity of these components (Non-Patent Document 11).
Weakly anionic polycarboxylic acid polymers have also been studied. Said polymers exhibit water solubility under physiological conditions (pH 7.4) but show hydrophobicity in a weakly acidic range (pH 5-6.8) which corresponds to the pH in the endosome, and thus disrupt the endosomal membrane, thereby enabling release of a gene/drug from the endosome into the cytoplasm. Those known anionic polyerms include poly(ethyl acrylic acid) (Non-Patent Document 12) and poly(propyl acrylic acid) (Non-Patent Document 13), and it was shown that a polycarboxylic acid polymer having pKa of about 5 is less cytotoxic and effective for release of a nucleic acid from the endosome. Other reports stated that succinylated polyglycidol (Non-Patent Document 14), and pseudo-peptides prepared by modifying the side chains of poly(L-lysine iso-phthalamide) with L-phenylalanine (Non-Patent Document 15) are effective as pH-responsive anionic polymers in release of genes/drugs from the endosome, more specifically uptake of endocytosed drugs into the cytoplasm.
Examples of the modifications of carboxy in hyaluronic acid with amino acids include modification with a glycine ethyl ester using, as a condensing agent, 4-(4,6-dimethoxy-1,3,5-triazine)-4-methylmorpholinium (hereinafter also referred to as DMT-MM) formed with 2-chloro-4,6-dimethoxy-1,3,5-triazine in the presence of N-methylmorpholine, but the degree of this modification was 20% at the maximum (Non-Patent Document 16). Examples of the modifications using triazine compounds as a condensing agent, which were reported in the documents disclosed after the priority date of the present application, include alanine introduced hyaluronic acid (Non-Patent Document 18); and modifications with other amino acids by the same procedures were also reported (Non-Patent Document 19, Patent Document 10). There was yet another report stating that hyaluronic acid was modified with leucine methyl ester hydrochloride, valine methyl ester hydrochloride, isoleucine methyl ester hydrochloride, proline methyl ester hydrochloride, phenylalanine methyl ester hydrochloride, arginine methyl ester hydrochloride, or histidine methyl ester hydrochloride using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter also referred to as EDC) as a condensing agent, and the resulting product was gelated without being deprotected, whereby a water-insoluble biocompatible film was prepared, but the degree of this modification was unknown (Patent Document 9).
As for modification of hyaluronic acid by amidation of carboxyl groups in hyaluronic acid with an aliphatic amine or an arylaliphatic amine, there were reported examples where benzylamine, octylamine, dodecylamine, and hexadecylamine were introduced using 1,1-carbonyldiimidazole in yields of 60%, 25%, 15% and 5%, respectively, but the degrees of these modifications were 60% at the maximum (Patent Document 11).